WO2002097446A1

WO2002097446A1 - Sample collection and preparation apparatus

Info

Publication number: WO2002097446A1
Application number: PCT/AU2002/000655
Authority: WO
Inventors: Chau Hoang Thanh Nguyen; William Samuel Hunter
Original assignee: Proteome Systems Intellectual Property Pty Ltd; Shimadzu Corporation
Priority date: 2001-05-25
Filing date: 2002-05-27
Publication date: 2002-12-05
Also published as: AUPR527201A0

Abstract

A sample manipulation apparatus for excising biomolecules such as proteins from a 2D gel includes a control means (48), a scanner means (14) including means for receiving an array of samples (24) and imaging means for generating an electronic image of the array, and a cutting tool (28) for removing a specified part of the array under the control of the control means (48). The apparatus is enclosed in a casing (40) and further includes the facility for automatically processing and handling the sample excised from the gel inside the casing including plates for receiving excised biomolecules, reagents and means for delivering liquid reagents (30) to the samples. The apparatus may also include sample incubation means. Drawers (23, 24) allow gels and reagents to be loaded into the apparatus with the reduced risk of contamination and the control means (48) also facilitates automatic operation of the apparatus with the gels and reagents loaded into the apparatus automatically on the stacker means (60).

Description

Sample collection and preparation apparatus

Field of the Invention

The present invention relates to a sample collection and preparation apparatus. In particular, the invention relates to an automated sample excision and preparation system for excising biomolecules (typically proteins) from an array of biomolecules. Typically the array will be carried in a gel or on a membrane.

Background of the Invention

Improvements in laboratory techniques and practices have led to the discovery of an ever increasing number of new biomolecules. New protein purification and detection methods, for example, have allowed the detection of many possibly new proteins. Due to the large number of known biomolecules, it is now necessary to carry molecular comparisons of newly discovered molecules to determine to what extent they are similar to, or different from, known molecules. To carry out definitive analyses for proteins for example it is necessary to obtain amino acid sequence information. Unfortunately, current methods and apparatus for such analyses are slow and are only able to analyse one or at best a few samples, at any one time. In order to carry out analysis of a given protein at present it is necessary to obtain the protein in substantially pure and isolated form.

It is known to use 2D PAGE gel electrophoresis to separate a sample of biomolecules into an array of separate "spots". The separate spots can then be removed from the array and analysed by adding reagents or the like. At first, the spots of biomolecules of interest were excised from the gel by hand using a scalpel or the like. However, such a manual process is slow and laborious and unsuited to high-throughput methods which are required to analyse the every increasing number of biomolecules which are being discovered. Thus, attempts have been made to automate the cutting and excision process.

US 5587062 to Shimadzu Corporation, Japan discloses an apparatus for automatically or manually collecting specified sections of a biomolecular sample which has been separated by gel electrophoresis. The apparatus comprises a base table, a cutting tool disposed above the base table, an X-Y moving mechanism for moving the cutting tool above the base table, a mechanism for moving the cutting tool up and down in the Z axis, a spot detector and a control device. A gel in the form of a slab is placed on the base table in such a way that the exposed gel faces upwards. The cutting tool defines a tubular cutting head which can be used to excise one or more spots from the gel by pressing the cutting tool into the gel. The spot detector operatively detects migration patterns in the gel placed on the base table. The control device controls the motion of the moving mechanism so as to have a portion of the gel at a specified migration band automatically or manually cut out by the cutting tool. Although the apparatus disclosed in US 5587062 is a considerable improvement on manual excision of spots in gels using a scalpel, there are many hundreds of thousands of proteins yet to be analysed to determine their properties, potential uses, and to what extent they are similar to or different from known molecules. The recent upsurge in interest in the field of proteomics means that an ever increasing number of new proteins are being discovered and there is a need to analyse biomolecules such as proteins in a more efficient automated way which allows for a high throughput of samples.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application. It is an object of the present invention to provide an improved sample cutting and preparation system for handling and analysing arrays of samples.

Summary of the Invention

In a first broad aspect the present invention provides a sample manipulation apparatus including: a control means; a scanner means including means for receiving an array of biomolecules and imaging means for generating an electronic image of the array; a cutting means for removing a specified part of the array under the control of the control means; and means for processing and handling the sample excised from the gel. The means for processing and handling the excised samples may include means for delivering liquid reagents onto the sample, either in situ in the array or after excision when the sample may be held in a receptacle such as a microtitre plate (MTP) inside the apparatus or the like for digestion or processing of the excised sample.

The apparatus may further include sample incubation means including heating means and temperature regulation means.

The means for handling may include means for loading a processed sample onto a MALDI plate of the like for subsequent MALDI-TOF (Matrix- Assisted Laser Desporption lonisation - Time of Flight Mass Spectrometry) analysis.

It is preferred that the apparatus is enclosed or sealed in the casing to reduce or inhibit contamination. The casing may include removable or openable transparent windows. Most typically the array of biomolecules will be present in a 2D gel however, they may be carried on a solid support, such as a membrane. The solid support can be a membrane made of polyvinylidene difluoride, nitrocellulose, nylon, teflon, zitex, polypropylene, PTFE, and derivatised forms thereof having one or more functional groups. Preferably, the biomolecules will be visualised by association with a dye, fluorescent group or metal, or by association with a second biomolecule which is coupled with a third biomolecule, dye, fluorescent group or metal.

In a preferred form, the samples are biomolecules selected from the group consisting of proteins, peptides, saccharides, lipids, nucleic acid molecules, complex biomolecules including glycoproteins, and mixtures thereof. The biomolecules are typically separated by chromatography to form an array of samples. The chromatography is preferably electrophoresis, and more preferably electrophoresis is carried out in a polyacrylamide gel. The polyacrylamide gel electrophoresis can be carried out in one dimension including isoelectric focusing, native polyacrylamide gel electrophoresis, and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Alternatively, the polyacrylamide gel electrophoresis is carried out in two dimensions with the first dimension by isoelectric focusing and the second dimension is by SDS polyacrylamide gel electrophoresis. In a preferred embodiment, drawer means are provided for loading gels or membranes into the apparatus. The loading of the gel via drawers, and the sealing of the instrument, also makes the machine safer to use and reduces the risk of contamination of the samples. This improves the ergonomics of the apparatus and makes it easier to load and use.

It is preferred that a further drawer is provided for loading reagents and other consumables into the apparatus.

In a particularly preferred embodiment, the loading of and unloading of sample arrays into the apparatus can be automated, the sample arrays being automatically fed into the apparatus, samples removed and analysed by the apparatus, with the "used" arrays being automatically removed from the machine from the opposite side of the apparatus from which they were loaded.

A stacker means for supporting a stack of arrays one above the other may be provided at the entry and exit to the apparatus. The loading drawer may or may not be removed from the apparatus to facilitate the feeding of arrays into the apparatus. A stacker means and automatic feeder may also be provided for automatically feeding consumables, such as reagents and the like into the apparatus and for removing waste disposables and other unwanted matter from the apparatus. The consumables and waste may be carried on trays to facilitate this and to allow stacking in order to keep the apparatus compact. The scanner may include a grey scale card to allow for the measuring of absolute intensity values of samples in the array.

The control means may be arranged to perform cutting based on detected fluorescent spectra to allow "blind" cutting of samples.

In a related aspect of the present invention, there is provided a method of sampling and treating one or more biomolecules present in a 2D array comprising the steps of: a. in a single integrated apparatus, making a computer readable image of a two dimensional array of biomolecules; b. excising a biomolecule from the array; c. treating the biomolecules with a reagent; d. analysing the treated biomolecule or preparing the biomolecule for analysis.

The step of preparing the biomolecule for analysis may include the step of placing the sample on a plate for MALDI-TOF analysis. Brief Description of the Drawings

A specific embodiment of the invention will now be described, by way of example only, and with reference to the accompanying drawings in which:

Figure 1 is a perspective view of an apparatus embodying the present invention;

Figure 2 shows the apparatus of Figure 1 with access panels opened;

Figure 3 shows the apparatus of Figure 1 with its cover removed;

Figure 4 shows the apparatus of Figure 3 with a top cover removed, and drawers open; Figure 5 illustrates the motion system of the apparatus of Figure 1 ;

Figure 6 is a rear view of the motion system;

Figure 7 is an underneath view of the motion system;

Figure 8 is a view of the apparatus of Figure 1 including automated loading and stacker means for automatic feeding of arrays and consumables into the apparatus;

Figure 9 illustrates calibration of the scanner;

Figure 10 is a schematic view of the control system of the apparatus; and

Figure 11 illustrates a typical database record of an array.

Detailed Description of a Preferred Embodiment

Referring to the drawings, the apparatus generally indicated at 10 includes a base frame or chassis 12 best seen in Figure 5, on which the apparatus is supported. A scanner 14 is supported on the base frame 12. The scanner 14 may be a high resolution "desktop" type scanner and includes a glass table 16 above which a two dimensional array of biomolecules such as proteins carried in a gel, or supported on a membrane can be placed. As is normal in a desktop type scanner, a scanning head traverses underneath the glass plate making an electronic image of that which is placed on or above the glass table. The image may be obtained by reflectance wherein a light source is located underneath the glass plate and the scanning head records light reflected by the gel. Alternatively, the image may be obtained in a transmissive scanning mode in which the light source is located above the glass plate and light is received by the scanning head after it has passed through the gel. With reference to Figure 9, the scanner table defines four permanent registration marks 18 known as fiducials, one in each corner of the scanner. A "grey scale" card 20 is also defined on the scanner table. These features allow the scanner to be calibrated as is described in more detail below.

As is best seen in Figure 2, the apparatus includes drawers 22a, 22b. One drawer includes a recess or gel tray 24 adapted to receive a gel containing an array of biomolecules. Gel tray adaptors 26 may be used to handle gels having different sizes. Up to three gels may be handled at one time depending on the size of the gels and the configuration of the handler. The other side of the drawer contains consumables. These may include trays 23 of disposable pipettes called "tips" and "zip-tips", trays of solvents or washing liquids 24, 96 or 384 well MTPs, MALDI target plates etc.

As is best seen in Figure 5, mounted for movement above the scanner, is a machine head 26 including a cutting tool 28 defining a cutting tip 28a, and an eight channel liquid delivery means 30 including eight adjacent liquid delivery tips 31. The machine head moves along an X axis 32 which in turn extends between and is supported by two Y axes 34a, 34b. The machine head includes a Z axis driver 36 for driving the cutting tool and liquid delivery system up and down in a vertical direction. Thus, the machine head can move not only in mutually perpendicular horizontal X and Y directions parallel to the surface of the base frame, but can also move in the vertical Z direction so that a specified portion of an array supported on the glass table 16 can be cut out and collected by the cutting tool 28. The machine head may also be moved so that the liquid delivery tips are positioned to dispense reagents either onto samples "in situ" in the array, or, more usually, into microtitre plates containing the excised samples. Also shown in Figure 5, is a cover plate 38 for covering the gels positioned above the scanner, and an actuator 39 for moving the cover plate. Figure 5 also illustrates one rail 80 of a rail system comprising two parallel spaced apart rails 80 and 81 (refer to Figure 6), and a drawer roller slider 82 which slides along the rail 80 facilitating the sliding of the drawer in and out of the apparatus.

As is best seen in Figures 1 and 2, the apparatus is enclosed in a casing or enclosure 40 including a base moulding 42 and a top moulding 44. The casing protects the apparatus from contamination. One end wall 46 of the casing folds down to allow access to a computer control unit 48 of the apparatus. Two sections 50, 52 of the top of the casing are transparent. The two sides 54, 56 of the casing are also transparent and are slidably located in the casing 44 such that they can be raised as shown in Figure 2, to allow access to the interior of the casing when required. Also shown in Figures 1 and 2 are caps 58 for reservoirs which contain purified water for operating the liquid delivery means 30. The liquid delivery means of this apparatus is described in more detail in applicants' co-pending International patent application "Liquid handling means for excision apparatus" filed 27 May 2002, the entire contents of which are incorporated herein by reference.

Figure 8 shows the apparatus of Figure 1 modified for high throughput analysis of sample arrays. In this version, the transparent side walls 54, 56 are removed and automated stacking and feeding means 60 are attached to the apparatus and loaded with banks of sample arrays, stacked one on top of another and trays of consumables which are loaded into the apparatus as required. The operation is controlled by the control means 60. The "used" sample arrays which have been processed and used consumables exit the apparatus from the opposite side from which they entered, therefore enabling ultra high throughput samples through the apparatus.

Figure 10 illustrates the control system for the apparatus which includes the embedded computer/control unit 48 which operates X, Y, Z actuators of the motion control means as well as actuators associated with the liquid-handling and delivery means. An external computer console 62 is linked to the embedded computer 48 for controlling the same externally. The external computer console 62 or computer 48 may be used to control the. automated stacking and feeding means 60.

In use, the operator first calibrates the scanner by placing a calibration gel 64 in the drawer and loading it into the apparatus so that it is located above the scanner table 16. The control means is programmed to automatically carry out a calibration process on instruction and directs the cutting head 28 to cut five spots 65 in the pattern shown in Figure 9. From the scanned positions of the fiducials and the cut spots, the control system can calculate interpolation functions which will enable it to position the cutting head 28 accurately above a given location on the gel. The grey scale 20 also enables calibration of absolute intensity of images. This process is described in more detail in one of the co-applicant's co-pending International patent application entitled "Imaging means for excision apparatus" filed 27 May 2002, the contents of which are incorporated herein by reference. Next, the operator mounts a stained 2D gel over the glass table via the drawer 22a. The internal scanner scans the gel. Image processing software identifies and catalogues spots on the gel to be analysed. The control means operates the cutting tool 28 to cut and extract samples and loads them onto microtitre plates (MTP) which have 96 (8 12) wells.

The remaining steps are handled eight at a time by the bank of dispensing tips, associated with the liquid handling means 30 i.e. the tips handle one row of the microtitre plate at a time.

The samples are cleaned of stain, desalted and digested. This is done by the apparatus positioning the probes successively over the MTP and reagent solutions. By a combination of dispense and aspirate operations with the liquid handling means picking up and dispensing reagents, the liquid handling means washes the samples, desalts and digests them, and loads the digested samples in the form of peptides into a MTP. Plain or resin treated pipette tips (zip tips) are loaded and ejected from the liquid handling means as required.

Next the treated sample is mixed with matrix, positioned over a MALDI target plate and deposited so that the samples ready for mass spectrometry. When all the samples deposited are dry, the operator may remove the target plate via the drawer and proceed immediately to mass spectrometry.

Detailed Example of Operation Session preparation

1. The operator prepares stained 2D gel/s onto shallow transparent tray/s. Depending on gel size the instrument is capable of accepting three gel trays

2. Consumables gets loaded on the drawer 22b, i.e. pipette tips, zip-tips, cleaning, digestion solutions, MTPs, and MSTP.

3. The gel tray is loaded onto the scan drawer 22a. Depending on gel sizes the scan drawer would have been configured with the correct gel tray adaptor. 4. All drawers close and session programming begins. Sample Excision

5. An internal optical scanner scans the gel

6. Internal image processing software identifies and catalogues spots on the gel to be analysed. The software converts pixel location into instrument co- ordinates. 7. The robot cutter on an overhead gantry move and position over each spots and accurately cuts samples with a twirl/vibrating/twist motion.

8. The cutting head 28 aspirate to extract the gel spot from the gel

9. The cutting head the move over to an available well on one of the MTPs and dispenses the sample.

Sample Separation

10. After spots are excised and delivered into the MTP wells, the 8 pipette probes add 100 μl of de-staining solution onto the samples, aspirate and repeat if necessary 11. Probes add 100 μl of dehydration solution, aspirate

12. Probes add enzyme solution (incubate overnight)

13. Probes add extraction solution (sonicate off-line)

14. Probes reduce organic solvent content by adding water. If the vacuum units are present this step is done by vacuum. Sample Transfer

15. Probes concentrate samples onto Zip-Tip by multiple aspirate and dispense strokes of the W-axis, 8 wells at a time.

16. Probes elute the peptides from the Zip-Tip using 2-3μl of matrix extraction solution onto the MSTP (MALDI TARGET PLATE). 17. Probes ejects the Zip-Tips into the tip bins, reload a fresh row of Zip-Tips and restart previous two steps.

18. After MSTP is full the samples are ready to go into the mass spectrometer.

The process outlined above, is completely automated from the loading of the gel to removal of the MALDI target plate and may be run without intervention by the operator.

However, the operator may select which spots to cut out or re-cut, if one or more of the spots are missed by the cutting tool.

Digestion may be performed off-line, freeing the apparatus up for other incision/deposition operations.

Additional steps such as sonication, incubation, vacuum drying may be inserted at any stage in the procedure.

The information relating to each array including the location and intensity of spots in the array is stored in a database and typical database structure is illustrated in Figure 10. Each gel may be impregnated with a RF tag for identification. Each gel has its own record, and the information stored includes project, experiment and gel name. The apparatus stores a complete set of spot locations, with intensities in the database.

Advantages of the Invention The automatic in feed of samples and consumables into the apparatus and the automatic retrieval of used arrays and consumables allows the apparatus to operate with a very high throughput of sample arrays.

The use of an optical scanner 14 to carry out in-place imaging of the arrays allows the measurement of both relative and absolute intensity readings and obviates the need for removing image capture means traversing the array. The present invention thus integrates the process of capturing and analysing an image of an array of samples, cutting a sample from that array, processing and handling that sample and delivering liquid reagents to the sample within the apparatus. The apparatus includes an embedded computer control system which has network capability and includes a sample preparation and experimental results database which may be capable of integrating with other databases and database services.

In the manual configuration illustrated in Figure 1 , the Figure may be loaded with samples and consumables via the sliding drawers which negates the need for operators to reach into the work space of the apparatus which has adverse safety and contamination aspects. In one variant, the apparatus could include means for embedding an RF tag into the gel sample so that the system will automatically read the description of the gel sample associated with that RF tag together with its previous process and history.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

1. A sample manipulation apparatus including: a control means; a scanner means including means for receiving an array of samples and imaging means for generating an electronic image of the array; a cutting means for removing a specified part of the array under the control of the control means; and means for processing and handling the sample excised from the gel.

2. A sample manipulation apparatus as claimed in claim 1 wherein the samples are biomolecules and the means for processing and handling the excised samples includes means for delivering liquid reagents onto the samples.

3. A sample manipulation apparatus as claimed in claim 1 or claim 2 further including sample incubation means including heating means and temperature regulation means.

4. A sample manipulation apparatus as claimed in any preceding claim wherein the means for processing and handling the sample include means for loading a processed sample onto a MALDI plate

5. A sample manipulation apparatus as claimed in any preceding claim wherein the apparatus is enclosed or sealed in a casing.

6. A sample manipulation apparatus as claimed in claim 5 wherein the casing includes removable or openable transparent windows.

7. A sample manipulation apparatus as claimed in any preceding claim wherein a drawer means is provided for an array of samples into the apparatus.

8. A sample manipulation apparatus as claimed in claim 7 wherein a further drawer is provided for loading reagents and other consumables into the apparatus.

9. A sample manipulation apparatus as claimed in any preceding claim wherein the loading of and unloading of sample arrays into the apparatus is automated, with the sample arrays being automatically fed into one side of the apparatus, samples being removed and analysed by the apparatus, and with the "used" arrays being automatically removed from the machine from the opposite side of the apparatus from the one side from they were loaded.

10. A sample manipulation apparatus as claimed in claim 9 wherein a stacker means for supporting a stack of arrays one above the other is provided at the entry and exit to the apparatus.

11. A sample manipulation apparatus as claimed in claim 10 wherein a stacker means and automatic feeder are provided for automatically feeding consumables, such as reagents and the like into the apparatus on trays and for removing trays containing unwanted matter from the apparatus.

12. A sample manipulation apparatus as claimed in any preceding claim wherein the scanner includes a grey scale card to allow for the measuring of absolute intensity values of samples in the array.

13. A method of sampling and treating one or more biomolecules present in a 2D array comprising the steps of: a. in a single integrated apparatus, making a computer readable image of a two dimensional array of biomolecules; b. excising a biomolecule from the array; c. treating the biomolecules with a reagent; d. analysing the treated biomolecule and/or preparing the biomolecule for analysis.

14. A method of sampling and treating one or more biomolecules wherein the step of preparing the biomolecule for analysis may include the step of placing the sample on a plate for MALDI-TOF analysis.

15. A method as claimed in claim 13 or 14 wherein the step of treating the biomolecules with a reagent takes place with the biomolecule in situ in the array.

16. A method as claimed in claim 13 or 14 wherein the step of treating the biomolecules with a reagent takes place after excision of the biomolecule when the excised biomolecule is contained in a receptacle inside the apparatus. 17 A method as claimed in any one of claims 13 to 16 wherein the array of biomolecules is present in a 2D gel 18 A method as claimed in any one of claims 13 to 17 wherein the biomolecules are selected from the group consisting of proteins, peptides, saccharides, lipids, nucleic acid molecules, complex biomolecules including glycoproteins, and mixtures thereof.